This invention relates to an immunohistochemical method for staining histology or cytology specimens using non-enzymatic reagents which, nevertheless, provide a means for amplifying the detection of bound antibody to a cellular component by converting a plurality of molecules of a soluble chromogen to an insoluble chromophore, which deposits at the site of the component.
Histochemical techniques were developed to permit selective staining of particular cellular components, e.g., molecular species, in tissue or other histological specimens by virtue of unique physicochemical properties thereof. Immunohistochemical methods take advantage of the ability of specific antibodies to bind to cellular components and to other antigens, e.g., viral, bacterial, fungal or parasitic antigens and/or products of cells or microorganisms. The antibodies are tagged with labels to render them detectable, e.g., radioisotopes, fluorescent compounds, chromophores, enzymes and the like. The use of enzymes has the further advantage that it serves to amplify the detection reaction by catalyzing the conversion of many substrate molecules to produce molecules which, in turn, serve to promote the conversion of many molecules of a soluble chromogen to insoluble chromophores which then precipitate at the site of binding of the specific antibody.
Further modifications of this technique have used enzymes conjugated to second antibodies, which bind to the primary antibodies that recognize the cellular components. Other modifications have been primary antibodies conjugated to biotin or avidin, with enzymes conjugated to the other member of this pair. Such enzyme-linked staining techniques and reagents are reviewed in Primus et al., "Functional Histopathology of Cancer: A Review of Immunoenzyme Histochemistry", In Methods of Cancer Research, 20, 139-182 (Academic Press, New York, N.Y., 1982), and are familiar to the skilled artisan in this field.
The most commonly used enzymes in this technique have been peroxidases, glucose oxidase and phosphatases. Peroxidases typically operate by using hydrogen peroxide to oxidize a iron porphyrin bound to the enzyme. The oxidized cofactor in turn oxidizes a soluble chromogen, typically diaminobenzidine (DAB), aminoethylcarbazole, 4-chloro-1-naphthol, tetramethyl-benzidine or phenylenediamine/pyrocatechol, to form an insoluble dye, which deposits at the site of bound antibody to which the enzyme is directly or indirectly bound or linked through one or more bridging antibodies or other specific binding couples, e.g., an avidin-biotin couple.
Glucose oxidase typically operates by using a substrate, normally glucose, to reduce a bound cofactor, e.g., NAD+, which in turn reduces an electron transfer agent, e.g., phenazine methosulfate (PMS), and is itself reoxidized. The reduced electron transfer agent then reduces a soluble chromogen, e.g., a tetrazolium salt, to an insoluble dye, e.g., a formazan, which deposits at the site of bound antibody. In this variant, the electron transfer agent is added in soluble form and mediates electron transfer from the redox cofactor of the enzyme to the soluble chromogen.
Phosphatases typically operate by hydrolyzing a substrate which is a phophate ester of a substituted naphthol, e.g., naphthol AS phosphate (3-hydroxy-2-naphthoic acid anilide phosphate). The free napthol then reacts with a stable solubel diazotate in the developing solution, e.g, Fast Blue or Fast Red, forming an insoluble dye.
Another powerful advance involves the use of soluble immune complexes of the enzyme, e.g., a peroxidase/anti-peroxidase (PAP) complex, by which the enzyme is bound to the site of bound specific antibody through the intermediacy of a bridging antibody. Typically, the PAP complex uses antibodies of the same species as the primary antibody, and the bridging antibody is an anti-species antibody from another animal. For example, if the primary antibody and the anti-peroxidase are both murine antibodies, the bridging antibody could be goat anti-mouse IgG. This techniques has the advantage that it does not require covalent binding of the enzyme to an antibody or to another carrier or hapten.
However, enzymes have relatively limited stabilities, and they are stable over a relatively narrow range of conditions. In contrast, immunoglobulins are far more stable. Thus, an immunohistochemical reagent that contains an enzyme component has a limited shelf life primarily because of the presence of the enzyme. Furthermore, the fragility of the enzyme could limit the conditions under which it can be coupled to antibody or other linker.
A need therefore continues to exist for immunohistochemical reagent systems which embody the amplification which enzymes can impart, but which are enzyme-free and have longer shelf life and greater stability.